Bottom Line:
Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored.Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid.These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

Affiliation: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK.

ABSTRACTInositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

RSOB150014F5: Absence of InsP6 in human urine. Freshly collected urinefrom healthy anonymous donors (D1–D3) was centrifuged toremove any epithelial cells. The samples were divided into twoaliquots (25 ml each for D1 and D2, 10 ml for D3), EDTA was addedand InsP6 (2 nmol) was supplemented into the spiked(InsP6) aliquots. The samples were then PA extractedand subjected to TiO2 enrichment. The extracted inositolphosphates were resolved by PAGE and visualized with toluidine bluestaining. The gel presented is representative of three experimentalrepeats.

Mentions:
Since large volumes of acidified fluid can be subject to TiO2 beadextraction, this gave us the opportunity to assay InsP6 in biofluids.Initially, we used commercially available serum from bovine, equine and humansources. We extracted 20 ml of serum with TiO2 beads and analysed theextracts by PAGE. While we were able to detect an almost completeInsP6 recovery in the spiked samples, we did not recover anyInsP6 in non-spiked serum (electronic supplementary material,figure S2A,B). We next analysed human plasma from a commercial source. Similarto serum, TiO2 extraction and PAGE analysis showed thatInsP6 could not be recovered from non-spiked samples of humanplasma (figure4a). The lower limit of InsP6 standarddetection on PAGE is about 0.25 nmol (figures 4a,b and 5), thereforeTiO2-extracting 20 ml of plasma with a recovery of approximately85% (figure1e) indicates that the lower limit of plasmaInsP6 we are able to extract and detect is approximately 15 nM.Consequently, we conclude that substantially less than 15 nM InsP6 ispresent in human plasma, in agreement with the enzymatic radio-assay previouslyreported [8]. Figure 4.

RSOB150014F5: Absence of InsP6 in human urine. Freshly collected urinefrom healthy anonymous donors (D1–D3) was centrifuged toremove any epithelial cells. The samples were divided into twoaliquots (25 ml each for D1 and D2, 10 ml for D3), EDTA was addedand InsP6 (2 nmol) was supplemented into the spiked(InsP6) aliquots. The samples were then PA extractedand subjected to TiO2 enrichment. The extracted inositolphosphates were resolved by PAGE and visualized with toluidine bluestaining. The gel presented is representative of three experimentalrepeats.

Mentions:
Since large volumes of acidified fluid can be subject to TiO2 beadextraction, this gave us the opportunity to assay InsP6 in biofluids.Initially, we used commercially available serum from bovine, equine and humansources. We extracted 20 ml of serum with TiO2 beads and analysed theextracts by PAGE. While we were able to detect an almost completeInsP6 recovery in the spiked samples, we did not recover anyInsP6 in non-spiked serum (electronic supplementary material,figure S2A,B). We next analysed human plasma from a commercial source. Similarto serum, TiO2 extraction and PAGE analysis showed thatInsP6 could not be recovered from non-spiked samples of humanplasma (figure4a). The lower limit of InsP6 standarddetection on PAGE is about 0.25 nmol (figures 4a,b and 5), thereforeTiO2-extracting 20 ml of plasma with a recovery of approximately85% (figure1e) indicates that the lower limit of plasmaInsP6 we are able to extract and detect is approximately 15 nM.Consequently, we conclude that substantially less than 15 nM InsP6 ispresent in human plasma, in agreement with the enzymatic radio-assay previouslyreported [8]. Figure 4.

Bottom Line:
Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored.Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid.These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.

Affiliation:
Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK.

ABSTRACTInositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.